Polynucleotides encoding rodent antibodies with human idiotypes and animals comprising same

ABSTRACT

The invention relates to polynucleotides, particularly chimeric polynucleotides useful for optimal production of functional immunoglobulins with human idiotypes in rodents. The invention further relates to rodents comprising such polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Application No.PCT/US2013/075157 filed Decmeber 13, 2013, which claims priority to U.S.provisional patent application U.S. Ser. No. 61/737,371 filed 14 Dec.2012. The applications are expressly incorporated herein in theirentirety by reference.

FIELD OF INVENTION

The invention relates to polynucleotides, particularly chimericpolynucleotides useful for the production of immunoglobulins with humanidiotypes in rodents. The invention further relates to rodent cellscomprising such polynucleotides.

REFERENCE TO A SEQUENCE LISTING

A Sequence Listing is provided in this patent document as a txt fileentitled “189314-US_ST25.txt” and created Dec. 12, 2012 (size 3 MB). Thecontents of this file is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Human monoclonal antibodies have proven to be invaluable in therapeuticapplications, either as IgG of conventional size, single chains ordomain modules^(1, 2). Despite the successes there are still majorshortcomings in their production, which relies either on specificityselection of available human material and subsequent modification ofindividual products, or the immunization of the limited availability oftransgenic animals, mainly mice³. Target antigen restrictions are widelyin place for the use of transgenic mice, as well as large transgenicanimals such as cattle, and the development of new specificities iscompany controlled⁴⁻⁷.

DNA rearrangement and expression of human immunoglobulin (Ig) genes intransgenic mice was pioneered over 20 years ago by stably insertingheavy-chain genes in germline configuration⁸. Although human antibodyrepertoires were obtained in these early animals, major improvements,resulting in higher expression levels and exclusive production of humanIg, combined two new strategies: gene knock-out in embryonic stem (ES)cells⁹ and locus extension on artificial chromosomes¹⁰.

Silencing of the endogenous Ig genes by gene targeting in ES cellsproduced several inactive mouse lines without the ability to rearrangetheir IgH and Igκ locus or without producing fully functional IgH, Igκor Igλ, products (summarized in³). More recently zinc finger nucleases(ZFNs) were designed to generate site-specific double-strand breaks inIg genes, which allowed gene disruption by deletion and non-homologousDNA repair. Injection of ZFN plasmids into fertilized eggs produced Igsilenced rats and rabbits with IgH and IgL disruptions¹¹⁻¹³.

Efficient expression of antibodies requires functional regulatoryelements in various locations in immunoglobulin loci. Enhancer sequenceshave been identified near many active genes by nuclease digest andhypersensitivity to degradation. Hypersensitive sites may precedepromoter sequences and the strength of their activity was correlatedwith the DNA sequence. Linkage to reporter genes showed elevatedtranscription if enhancer function was present (Mundt et al., J.Immunol., 166, 3315[2001]. In the IgH locus two important transcriptionor expression regulators have been identified, Eμ and the 3′E at the endof the locus (Pettersson et al., Nature, 344, 165 [1990]). In the mousethe removal of the entire 3′ regulatory region (containing hs3a, hs1,2,hs3b and hs4) allows normal early B-cell development but abrogatesclass-switch recombination (Vincent-Fabert et al., Blood, 116, 1895[2010]) and possibly prevents the optimization of somatic hypermutation(Pruzina et al., Protein Engineering, Design and Selection, 1, [2011]).

The regulatory function to achieve optimal isotype expression isparticularly desirable when transgenic human IgH genes are being used.However, in a number of laboratories, transgene constructs with anincomplete 3′E region, typically providing only the hs1,2 element, ledto disappointing expression levels in transgenic mice even when theendogenous IgH locus was knocked-out. This may be one reason why thegeneration of antigen-specific fully human IgGs from geneticallyengineered mice has been inefficient thus far. (Lonberg et al., Nature368, 856 [1994]; Nicholson et al., J. Immunol., 163, 6898 [1999]; Daviset al., Cancer Metastasis Rev. 18, 421 [1999]; Pruzina et al., ProteinEngineering, Design and Selection, 1,[2011].

In the rat, the 3′E region has only been poorly analyzed. A comparisonof mouse and rat sequences does not allow identification of hs4, thecrucial 4th E element with additional important regulatory sequencesfurther downstream (Chatterjee et al., J. Biol. Chem., 286,29303[2011]). This could mean the region is not present in the rat, andperhaps not as important as in the mouse, or it could be absent in theanalyzed rat genome sequences.

Still needed are methods and materials for the optimal production ofimmunoglobulins or antibodies having human idiotypes using transgenicanimals, which are useful for treating humans in a broad range ofdisease areas.

SUMMARY OF INVENTION

Disclosed herein are novel polynucleotides comprising nucleic acidsequences encoding chimeric immunoglobulin chains, particularly chimericheavy chains for use in creating transgenic animals. The polynucleotidesof the present invention advantageously provide optimal expression due,at least in part, to the inclusion of a 3′ enhancer since translocilacking this 3′ enhancer result in impaired isotype switching and lowIgG expression. Accordingly, in preferred embodiments the inventionprovides chimeric polynucleotides comprising a rat 3′ enhancer sequence,an Ig constant region gene and at least one human immunoglubulin (Ig)joining (J) region gene. In a preferred embodiment, the rat 3′ enhancersequence comprises the sequence set forth as SEQ ID NO:1, or a portionthereof.

The chimeric polynucleotides set forth herein may further comprise atleast one human variable (V) gene, at least one a diversity (D) gene, ora combination thereof. In one embodiment, the constant region gene ofthe chimeric polynucleotide is selected from the group consisting of ahuman constant region gene and a rat constant region gene. In apreferred embodiment, the constant region gene is a rat constant regiongene. In another preferred embodiment, the constant region gene isselected from the group consisting of Cμ and Cγ.

In one embodiment, the chimeric polynucleotide comprises a nucleic acidsequence substantially homologous to the bacterial artificial chromosome(BAC) Annabel disclosed herein (e.g., SEQ ID NO:10), or a portionthereof, and may optionally further comprise at least one human variableIg gene isolatable from BAC6-V_(H)3-11 and BAC3. In a preferredembodiment, the chimeric polynucleotides contemplated herein comprisenucleic acid sequences (a) and (b) in 5′ to 3′ order: (a) a human Igvariable region comprising human V genes in natural configurationisolatable from BAC6-V_(H)3-11 and/or BAC3, and (b) a human Ig joiningregion comprising human J genes in natural configuration isolatable fromthe BAC Annabel. In another embodiment, each of the human Ig variableregion, human Ig diversity region, human Ig joining region, the Igconstant region and the rat 3′ enhancer region of a chimericpolynucleotide as disclosed herein are in the relative positions asshown in FIG. 1a . In another embodiment, a chimeric polynucleotide asdisclosed has a sequence comprising or substantially homologous to thesequence set forth as SEQ ID NO:2 or a portion thereof. In anotherembodiment, a chimeric polynucleotide as disclosed has a sequencecomprising or substantially homologous to the sequence set forth as SEQID NO:11, or a portion thereof. In a further embodiment, a chimericpolynucletoide as disclosed herein comprises a rearranged V-D-J regions,wherein said rearranged V-D-J regions encode a heavy chain variabledomain exon.

Also disclosed herein are polynucleotides encoding human kappa lightchain genes. In one embodiment, a polynucleotide as disclosed herein hasa nucleic acid sequence comprising or substantially homologous to anucleic acid sequence selected from the group consisting of RP11-1156D9(set forth as SEQ ID NO:3) and RP11-1134E24 (set forth as SEQ ID NO:4).In another embodiment, the isolated polynucleotide comprises nucleicacid sequences (a) and (b) in 5′ to 3′ order: (a) a human Ig variableregion comprising human V genes in natural configuration isolatable frombacterial artificial chromosomes (BAC) RP11-156D9 and/or RP11-1134E24;(b) a human Ig joining region comprising human J genes in naturalconfiguration isolatable from the bacterial artificial chromosomes (BAC)RP11-1134E24 and/or RP11-344F17 (set forth as SEQ ID NO:5). In apreferred embodiment, each of the human Ig variable region, the human Igjoining region, and the human Ig constant region are in relativeposition as shown in FIG. 1b . In another embodiment, a chimericpolynucleotide as disclosed has a sequence comprising or substantiallyhomologous to the sequence set forth as SEQ ID NO:6 or a portionthereof.

Also provided herein is a rodent cell comprising one or morepolynucleotides of the invention. For example, provided herein is arodent cell comprising a polynucleotide as disclosed herein, preferablycomprising a nucleic acid sequence encoding for a chimeric heavy chain,e.g., a nucleic acid sequence encoding a rat 3′ enhancer sequence, an Igconstant region gene and at least one human J region gene, andoptionally, comprising a nucleic acid sequence substantially homologousto the nucleic acid sequence selected from the group consisting ofRP11-1156D9, RP11-1134E24 and portions thereof. The rodent cellcontemplated herein may further comprise a polynucleotide encoding afunctional light chain, e.g., having a nucleic acid sequence comprisingor substantially homologous to a nucleic acid sequence selected from thegroup consisting of the sequence shown in FIG. 1b (set forth as SEQ IDNO:6), the sequence shown in FIG. 1c (set forth as SEQ ID NO:7), andportions thereof. In one embodiment, one or more of the polynucleotidesare integrated into the rodent cell genome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Integrated human Ig loci. (a) The chimeric human-rat IgH regioncontains 3 overlapping BACs with 22 different and potentially functionalhuman V_(H) segments. BAC6-3 has been extended with V_(H)3-11 to providea 10.6 kb overlap to BAC3, which overlaps 11.3 kb via V_(H)6-1 with theC region BAC Hu-Rat Annabel. The latter is chimeric and contains allhuman D and J_(H) segments followed by the rat C region with fullenhancer sequences. (b) The human Igk BACs with 12 Vks and all Jksprovide a ˜14 kb overlap in the Vk region and ˜40 kb in Ck to includethe KDE. (c) The human Igl region with 17 Vls and all J-Cls, includingthe 3′ enhancer, is from a YAC³³.

FIG. 2a-d : Flow cytometry analysis of lymphocyte-gated bone marrow andspleen cells from 3 months old rats. Surface staining for IgM and CD45R(B220) revealed a similar number of immature and mature B-cells in bonemarrow and spleen of HC14 and wt rats, while JKO/JKO animals showed noB-cell development. Plotting forward (FSC) against site (SSC) scattershowed comparable numbers of lymphocyte (gated) populations, concerningsize and shape. Surface staining of spleen cells with anti-IgG (G1, G2a,G2b, G2c isotype) plotted against cell count (×102) revealed near normalnumbers of IgG+ expressers in HC14 rats compared to wt. In FIG. 2a , Arefers to pro/pre B-cells (CD45R⁺IgM⁻) and B refers to immature B-cells(CD45R⁺IgM⁺). In FIG. 2c , A refers to transitional B cells(CD45R⁺IgM⁻), B to follicular B cells (CD45R⁺IgM⁺) and C to marginalzone B cells (CD45R^(low)IgM⁺).

FIG. 3: Mutational changes in IgH and IgL transcripts from PBLs. Unique(VHDJH)s and VLs were from amplifications with V group specific primers:IGHV1, 2, 3, 4 and 6 in combination with the universal γCH2 reverseprimer, IGLV2, 3 and 4 with reverse Cλ, primer; and IGKV1, 3, 4 and 5 inwith reverse Cκ primer (Supplementary Table 1). Mutated trans-switchproducts were identified for humanVH-rat Cγ2a (4) and human VH-rat Cγ2c(2).

FIG. 4: Purification of rat Ig with human idiotypes and comparison tohuman and normal rat Ig levels. OmniRat serum and human or rat wtcontrol serum, 100 μl each, was used for IgM/G purification. (a) IgM wascaptured with anti-IgM matrix, which identified 14 μg in wt rat, and 30μg and 10 μg in OmniRats [HC14(a) and HC14(c)]. (b) IgG was purified onprotein A and protein G columns, with a yield of up to ˜3 mg/ml forOmniRats (Protein A: HC14(a) 1000 μg/ml; HC14(b) 350 μg/ml; wt rat 350μg/ml; Protein G: HC14(a) 2970 μg/ml; HC14(b) 280 μg/ml; wt rat 1010μg/ml). (c) Human Igκ and (d) human Igλ was purified on anti-Igκ andanti-Igλ matrix, respectively. No purification product was obtainedusing wt rat serum (not shown). Purified Ig, ˜3 μg (concentrationdetermined by nano drop), was separated on 4-15% SDS-PAGE under reducingconditions. Comparison by ELISA titration of (e) human Igκ and (f) humanIgλ levels in individual OmniRats (8531, 8322, 8199, 8486, 8055), humanand wt rat serum. Serum dilution (1:10, 1:100, 1:1,000, 1:10,000) wasplotted against binding measured by adsorption at 492 nm. Matchingname/numbers refer to samples from the same rat.

DETAILED DESCRIPTION

Provided herein are chimeric polynucleotides encoding a recombinant orartificial immunoglobulin chain or loci. As described above, thechimeric polynucleotides disclosed herein are useful for thetransformation of rodents to include human Ig genes and for theproduction of immunoglobulins or antibodies having human idiotypes usingsuch rodents.

Polynucleotides

Immunoglobulin refers to a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes. Therecognized human immunoglobulin genes include the kappa, lambda, alpha(IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and muconstant region genes, as well as the myriad immunoglobulin variableregion genes. Full-length immunoglobulin “light chains” (about 25 Kd, or214 amino acids) generally comprise a variable domain encoded by an exoncomprising one or more variable region gene(s) and one or more joiningregion gene(s) at the NH₂-terminus (about 110 amino acids) and constantdomain encoded by a kappa or lambda constant region gene at theCOOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd,or 446 amino acids), similarly comprise (1) a variable domain (about 116amino acids) encoded by an exon comprising one or more variable regiongenes, one or more diversity region genes and one or more joining regiongenes, and (2) one of the aforementioned constant domains comprising oneor more constant region genes, e.g., alpha, gamma, delta, epsilon or mu(encoding about 330 amino acids). The immunoglobulin heavy chainconstant region genes encode for the antibody class, i.e., isotype(e.g., IgM or IgG1).

As used herein, the term “antibody” refers to a protein comprising atleast one, and preferably two, heavy (H) chain variable domains(abbreviated herein as VH), and at least one and preferably two light(L) chain variable domains (abbreviated herein as VL). An ordinarilyskilled artisan will recognize that the variable domain of animmunological chain is encoded in gene segments that must first undergosomatic recombination to form a complete exon encoding the variabledomain. There are three types of regions or gene segments that undergorearrangement to form the variable domain: the variable regioncomprising variable genes, the diversity region comprising diversitygenes (in the case of an immunoglobulin heavy chain), and the joiningregion comprising joining genes. The VH and VL domains can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDRs”) interspersed with regions that are moreconserved, termed “framework regions” (“FRs”). The extent of the FRs andCDRs has been precisely defined (see, Kabat et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; and Chothia etal. (1987) J. Mol. Biol. 196:901-17, which are hereby incorporated byreference). Each VH and VL domain is generally composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antigenbinding fragment of an antibody (or simply “antibody portion,” or“fragment”), as used herein, refers to one or more fragments of afull-length antibody that retain the ability to specifically bind to anantigen (e.g., CD3).

Examples of binding fragments encompassed within the term “antigenbinding fragment” of an antibody include (i) an Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii)an F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) an Fd fragmentconsisting of the VH and CH1 domains; (iv) an Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al. (1989) Nature 341:544-46), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they may be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see, e.g., Bird et al.(1988) Science 242:423-26; and Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-83). Such single chain antibodies are also intended tobe encompassed within the term “antigen binding fragment” of anantibody. These antibody fragments are obtained using conventionaltechniques known to those skilled in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An antibody may further include a heavy and/or light chain constantdomain to thereby form a heavy and light immunoglobulin chain,respectively. In one embodiment, the antibody is a tetramer of two heavyimmunoglobulin chains and two light immunoglobulin chains, wherein theheavy and light immunoglobulin chains are interconnected, e.g., bydisulfide bonds. The heavy chain constant domain is comprised of threegene segments, CH1, CH2 and CH3. The light chain constant domain iscomprised of one gene, CL. The variable domains of the heavy and/orlight chains contain a binding domain that interacts with an antigen.The constant domains of the antibodies typically mediate the binding ofthe antibody to host tissues or factors, including various cells of theimmune system (e.g., effector cells) and the first component (C1q) ofthe classical complement system.

By polynucleotide encoding an artificial immunoglobulin locus orartificial immunoglobulin chain is meant an recombinant polynucleotidecomprising multiple immunoglobulin regions, e.g., a variable (V) regionor gene segment comprising V genes, a joining (J) gene region or genesegment comprising J genes, a diversity (D) region or gene segmentcomprising D genes in the case of a heavy chain locus and/or at leastone constant (C) region comprising at least one C gene. Preferably, eachregion of the variable domain, e.g., V, D, or J region, comprises orspans at least two genes of the same type. For example a variable regionas used herein comprises at least two variable genes, a joining regioncomprises at least two joining genes and a diversity region comprisestwo diversity genes. A constant region may comprise only one constantgene, e.g. a κ gene or λ gene, or multiple genes, e.g., CH1, CH2, andCH3.

“Enhancer sequences” or “enhancer” as used herein refers to sequencesthat have been identified near many active genes by nuclease digest andhypersensitivity to degradation. Hypersensitive sites may precedepromoter sequences and the strength of their activity was correlatedwith the DNA sequence. Linkage to reporter genes showed elevatedtranscription if enhancer function was present (Mundt et al., J.Immunol., 166, 3315[2001]). In the IgH locus two important transcriptionor expression regulators have been identified, Eμ and the 3′E at the endof the locus (Pettersson et al., Nature, 344, 165 [1990]). In the mousethe removal of the whole 3′ regulatory region (containing hs3a, hs1,2,hs3b and hs4) allows normal early B-cell development but abrogatesclass-switch recombination (Vincent-Fabert et al., Blood, 116, 1895[2010]) and possibly prevents the optimization of somatic hypermutation(Pruzina et al., Protein Engineering, Design and Selection, 1, [2011]).The regulatory function to achieve optimal isotype expression isparticularly desirable when transgenic human IgH genes are being used.Transgene constructs with incomplete 3′E region, usually only providingthe hs1,2 element, led to disappointing expression levels in transgenicmice even when the endogenous IgH locus was knocked-out. As aconsequence, only few antigen-specific fully human IgGs have beenisolated from constructs produced in the last 20 years (Lonberg et al.,Nature 368, 856 [1994]; Nicholson et al., J. Immunol., 163, 6898 [1999];Davis et al., Cancer Metastasis Rev. 18, 421 [1999]; Pruzina et al.,Protein Engineering, Design and Selection, 1, [2011]). In the rat IgHlocus, the 3′E region has only been poorly analyzed. A comparison ofmouse and rat sequences did not allow identification of hs4, the crucial4^(th) element with additional important regulatory sequences furtherdownstream (Chatterjee et al., J. Biol. Chem., 286,29303 [2011]). Thepolynucleotides of the present invention advantageously provide optimalexpression due, at least in part, to the inclusion of a rat 3′ enhancersince chimeric polynucleotides lacking this 3′ enhancer result inimpaired isotype switching and low IgG expression. In one embodiment,the rat 3′ enhancer has a sequence comprising or substantiallyhomologous to the sequence set forth as SEQ ID NO:1 or a portionthereof.

As used herein, a polynucleotide having a sequence comprising orsubstantially homologous to a portion, e.g., less than the entirety, ofsecond sequence (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.) preferablyretains the biological activity of the second sequence (e.g., retainsthe biological activity of a 3′ enhancer to provide optimal expressionand/or isotype switching of immunoglobulins, is capable of rearrangementto provide a humanized chimeric heavy chain, etc.). In one embodiment, anucleic acid comprising a sequence comprising or substantiallyhomologous to a portion of SEQ ID NO:1 comprise at least 8 kB,preferably at least 10 kB of continuous nucleic acids that aresubstantially homologous to SEQ ID NO:1.

“Artificial Ig locus” as used herein may refer to polynucleotides that(e.g., a sequence comprising V-,D-, and/or J regions in the case ofheavy chain, or V- and/or J regions in the case of light chain, andoptionally a constant region for either or both a heavy and light chgin)that are unrearranged, partially rearranged, or rearranged. ArtificialIg loci include artificial Ig light chain loci and artificial Ig heavychain loci. In one embodiment, an artificial immunoglobulin locus of theinvention is functional and capable of rearrangement and producing arepertoire of immunoglobulin chains. In a preferred embodiment, thevariable domain or portion thereof of a polynucleotide disclosed hereincomprises genes in natural configuration, i.e., naturally occurringsequences of an human Ig gene segment, degenerate forms of naturallyoccurring sequences of a human Ig gene segment, as well as syntheticsequences that encode a polypeptide sequence substantially identical tothe polypeptide encoded by a naturally occurring sequence of a human Iggene segment. In another preferred embodiment, the polynucleotidecomprises a variable domain or portion thereof in a naturalconfiguration found in humans. For example, a polynucleotide encoding anartificial Ig heavy chain as disclosed herein may comprise in naturalconfiguration at least two human V genes, at least two D genes, at leasttwo J genes or a combination thereof.

In a preferred embodiment, an artificial Ig locus comprises a non-humanC region gene and is capable of producing a repertoire ofimmunoglobulins including chimeric immunoglobulins having a non-human Cregion. In one embodiment, an artificial Ig locus comprises a human Cregion gene and is capable of producing a repertoire of immunoglobulinsincluding immunoglobulins having a human C region. In one embodiment, anartificial Ig locus comprises an “artificial constant region gene”, bywhich is meant a constant region gene comprising nucleotide sequencesderived from human and non-human constant regions genes. For example, anexemplary artificial C constant region gene is a constant region geneencoding a human IgG CH1 domain and rat IgG CH2 and CH3 domain.

In a preferred embodiment, an artificial Ig locus comprises 3′ enhancersequences, including hs1,2, hs3a, hs3b and sequences (500-2500 nt)downstream of hs3b. In transgenic animals, artificial loci comprisingthe full ˜30 kb 3′E region from Calpha to 3′ hs3b result in high levelIgG expression, extensive hypermutation and large numbers ofantigen-specific hybridomas of high (pM) affinity. However, shorterenhancer sequences reduce Ig expression.

In a preferred embodiment, an artificial Ig locus comprises the 3′enhancer sequence shown in FIG. 1a . This sequence is derived from therat Ig heavy chain locus and contains about 30 kb of the 3′ region fromCalapha to 3′ hs3b. The sequence of the rat 3′ enhancer sequence is setforth as SEQ ID NO:1. In another embodiment, the artificial Ig locuscomprises a sequence comprising or substantially homologous to thesequence set forth as SEQ ID NO:1, or a portion thereof.

In some embodiments, an artificial Ig heavy chain locus lacks CH1, or anequivalent sequence that allows the resultant immunoglobulin tocircumvent the typical immunoglobulin: chaperone association. Suchartificial loci provide for the production of heavy chain-onlyantibodies in transgenic animals which lack a functional Ig light chainlocus and hence do not express functional Ig light chain. Suchartificial Ig heavy chain loci are used in methods contemplated hereinto produce transgenic animals lacking a functional Ig light chain locus,and comprising an artificial Ig heavy chain locus, which animals arecapable of producing heavy chain-only antibodies. Alternatively, anartificial Ig locus may be manipulated in situ to disrupt CH1 or anequivalent region and generate an artificial Ig heavy chain locus thatprovides for the production of heavy chain-only antibodies. Regardingthe production of heavy chain-only antibodies in light chain-deficientmice, see for example Zou et al., JEM, 204:3271-3283, 2007.

By “human idiotype” is meant a polypeptide sequence present on a humanantibody encoded by an immunoglobulin V-gene segment. The term “humanidiotype” as used herein includes both naturally occurring sequences ofa human antibody, as well as synthetic sequences substantially identicalto the polypeptide found in naturally occurring human antibodies. By“substantially” is meant that the degree of amino acid sequence identityis at least about 85%-95%. Preferably, the degree of amino acid sequenceidentity is greater than 90%, more preferably greater than 95%.

By a “chimeric antibody” or a “chimeric immunoglobulin” is meant animmunoglobulin molecule comprising a portion of a human immunoglobulinpolypeptide sequence (or a polypeptide sequence encoded by a human Iggene segment) and a portion of a non-human immunoglobulin polypeptidesequence. The chimeric immunoglobulin molecules of the present inventionare immunoglobulins with non-human Fc-regions or artificial Fc-regions,and human idiotypes. Such immunoglobulins can be isolated from animalsof the invention that have been engineered to produce chimericimmunoglobulin molecules.

By “artificial Fc-region” is meant an Fc-region encoded by an artificialconstant region gene.

The term “Ig gene segment” as used herein refers to regions of DNAencoding various portions of an Ig molecule, which are present in thegermline of non-human animals and humans, and which are brought togetherin B cells to form rearranged Ig genes. Thus, Ig gene segments as usedherein include V gene segments, D gene segments, J gene segments and Cgene segments.

The term “human Ig gene segment” as used herein includes both naturallyoccurring sequences of a human Ig gene segment, degenerate forms ofnaturally occurring sequences of a human Ig gene segment, as well assynthetic sequences that encode a polypeptide sequence substantiallyidentical to the polypeptide encoded by a naturally occurring sequenceof a human Ig gene segment. By “substantially” is meant that the degreeof amino acid sequence identity is at least about 85%-95%. Preferably,the degree of amino acid sequence identity is greater than 90%, morepreferably greater than 95%.

Polynucleotides related to the present invention may comprise DNA or RNAand may be wholly or partially synthetic. Reference to a nucleotidesequence as set out herein encompasses a DNA molecule with the specifiedsequence, and encompasses an RNA molecule with the specified sequence inwhich U is substituted for T, unless context requires otherwise.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). In a preferred embodiment,the length of a reference sequence aligned for comparison purposes is atleast 30%, preferably at least 40%, more preferably at least 50%, evenmore preferably at least 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent sequenceidentity between two sequences may be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-53) algorithm, which has been incorporatedinto the GAP program in the GCG software package (available online atgcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gapweight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6. In yet another preferred embodiment, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package (available at www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set ofparameters (and the one that should be used if the practitioner isuncertain about what parameters should be applied to determine if amolecule is within a sequence identity or homology limitation of theinvention) is a Blossum 62 scoring matrix with a gap penalty of 12, agap extend penalty of 4, and a frameshift gap penalty of 5. The percentidentity between two amino acid or nucleotide sequences can also bedetermined using the algorithm of Meyers and Miller ((1989) CABIOS4:11-17), which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

Artificial Ig Loci

The present invention is further directed to artificial Ig loci andtheir use in making transgenic animals capable of producingimmunoglobulins having a human idiotype. Each artificial Ig locuscomprises multiple immunoglobulin gene segments, which include at leastone V region gene segment, one or more J gene segments, one or more Dgene segments in the case of a heavy chain locus, and one or moreconstant region genes. In the present invention, at least one of the Vgene segments encodes a germline or hypermutated human V-region aminoacid sequence. Accordingly, such transgenic animals have the capacity toproduce a diversified repertoire of immunoglobulin molecules, whichinclude antibodies having a human idiotype. In heavy chain loci human ornon-human-derived D-gene segments may be included in the artificial Igloci. The gene segments in such loci are juxtaposed with respect to eachother in an unrearranged configuration (or “the germlineconfiguration”), or in a partially or fully rearranged configuration.The artificial Ig loci have the capacity to undergo gene rearrangement(if the gene segments are not fully rearranged) in the subject animalthereby producing a diversified repertoire of immunoglobulins havinghuman idiotypes.

Regulatory elements like promoters, enhancers, switch regions,recombination signals, and the like may be of human or non-human origin.What is required is that the elements be operable in the animal speciesconcerned, in order to render the artificial loci functional. Preferredregulatory elements are described in more detail herein.

In one aspect, the invention provides transgenic constructs containingan artificial heavy chain locus capable of undergoing gene rearrangementin the host animal thereby producing a diversified repertoire of heavychains having human idiotypes. An artificial heavy chain locus of thetransgene contains a V-region with at least one human V gene segment.Preferably, the V-region includes at least about 5-100 human heavy chainV (or “VH”) gene segments. As described above, a human VH segmentencompasses naturally occurring sequences of a human VH gene segment,degenerate forms of naturally occurring sequences of a human VH genesegment, as well as synthetic sequences that encode a polypeptidesequence substantially (i.e., at least about 85%-95%) identical to ahuman heavy chain V domain polypeptide.

In a preferred embodiment, the artificial heavy chain locus contains atleast one or several rat constant region genes, e.g., Cδ, Cμ and Cγ(including any of the Cγ subclasses).

In another preferred embodiment, the artificial heavy chain locuscontains artificial constant region genes. In a preferred embodiment,such artificial constant region genes encode a human CH1 domain and ratCH2 CH3 domains, or a human CH1 and rat CH2, CH3 and CH4 domains. Ahybrid heavy chain with a human CH1 domain pairs effectively with afully human light chain.

In a preferred embodiment, an artificial Ig locus comprises 3′ enhancersequences, including hs1,2, hs3a, hs3b and sequences between rat Calphaand 3′hs3b.

In another preferred embodiment, the artificial heavy chain locuscontains artificial constant region genes lacking CH1 domains In apreferred embodiment, such artificial constant region genes encodetruncated IgM and/or IgG lacking the CH1 domain but comprising CH2, andCH3, or CH1, CH2, CH3 and CH4 domains. Heavy chains lacking CH1 domainscannot pair effectively with Ig light chains and form heavy chain onlyantibodies.

In another aspect, the invention provides transgenic constructscontaining an artificial light chain locus capable of undergoing generearrangement in the host animal thereby producing a diversifiedrepertoire of light chains having human idiotypes. An artificial lightchain locus of the transgene contains a V-region with at least one humanV gene segment, e.g., a V-region having at least one human VL geneand/or at least one rearranged human VJ segment. Preferably, theV-region includes at least about 5-100 human light chain V (or “VL”)gene segments. Consistently, a human VL segment encompasses naturallyoccurring sequences of a human VL gene segment, degenerate forms ofnaturally occurring sequences of a human VL gene segment, as well assynthetic sequences that encode a polypeptide sequence substantially(i.e., at least about 85%-95%) identical to a human light chain V domainpolypeptide. In one embodiment, the artificial light chain Ig locus hasa C-region having at least one rat C gene (e.g., rat Cλ or Cκ).

Another aspect of the present invention is directed to methods of makinga transgenic vector containing an artificial Ig locus. Such methodsinvolve isolating Ig loci or fragments thereof, and combining the same,with one or several DNA fragments comprising sequences encoding human Vregion elements. The Ig gene segment(s) are inserted into the artificialIg locus or a portion thereof by ligation or homologous recombination insuch a way as to retain the capacity of the locus to undergo effectivegene rearrangement in the subject animal.

Preferably, a non-human Ig locus is isolated by screening a library ofplasmids, cosmids, YACs or BACs, and the like, prepared from the genomicDNA of the same. YAC clones can carry DNA fragments of up to 2megabases, thus an entire animal heavy chain locus or a large portionthereof can be isolated in one YAC clone, or reconstructed to becontained in one YAC clone. BAC clones are capable of carrying DNAfragments of smaller sizes (about 50-500 kb). However, multiple BACclones containing overlapping fragments of an Ig locus can be separatelyaltered and subsequently injected together into an animal recipientcell, wherein the overlapping fragments recombine in the recipientanimal cell to generate a continuous Ig locus.

Human Ig gene segments can be integrated into the Ig locus on a vector(e.g., a BAC clone) by a variety of methods, including ligation of DNAfragments, or insertion of DNA fragments by homologous recombination.Integration of the human Ig gene segments is done in such a way that thehuman Ig gene segment is operably linked to the host animal sequence inthe transgene to produce a functional humanized Ig locus, i.e., an Iglocus capable of gene rearrangement which lead to the production of adiversified repertoire of antibodies with human idiotypes. Homologousrecombination can be performed in bacteria, yeast and other cells with ahigh frequency of homologous recombination events. Engineered YACs andBACs can be readily isolated from the cells and used in makingtransgenic animals

Rodent Oocytes and Transgenic Animals Comprising Artificial Ig Loci andCapable of Producing Antibodies Having Human Idiotypes

In one aspect, the invention provides transgenic animals capable ofproducing immunoglobulins having human idiotypes, as well as methods ofmaking the same.

The transgenic animals used are selected from rodents (e.g., rats,hamsters, mice and guinea pigs).

The transgenic animals used for humanized antibody production in theinvention carry germline mutations in endogenous Ig loci. In a preferredembodiment, the transgenic animals are homozygous for mutated endogenousIg heavy chain and/or endogenous Ig light chain genes. Further, theseanimals carry at least one artificial Ig locus that is functional andcapable of producing a repertoire of immunoglobulin molecules in thetransgenic animal. The artificial Ig loci used in the invention includeat least one human V gene segment.

In a preferred embodiment, the transgenic animals carry at least oneartificial Ig heavy chain locus and at least one artificial Ig lightchain locus that are each functional and capable of producing arepertoire of immunoglobulin molecules in the transgenic animal, whichrepertoire of immunoglobulin molecules includes antibodies having ahuman idiotype. In one embodiment, artificial loci including at leastone non-human C gene are used, and animals capable of producing chimericantibodies having a human idiotype and non-human constant region areprovided. In one embodiment, artificial loci including at least onehuman C gene are used, and animals capable of producing antibodieshaving a human idiotype and human constant region are provided.

In another preferred embodiment, the transgenic animals carry at leastone artificial Ig heavy chain locus, and lack a functional Ig lightchain locus. Such animals find use in the production of heavy chain—onlyantibodies.

Production of such transgenic animals involves the integration of one ormore artificial heavy chain Ig loci and one or more artificial lightchain Ig loci into the genome of a transgenic animal having at least oneendogenous Ig locus that has been or will be inactivated by the actionof one or more meganucleases. Preferably, the transgenic animals arenullizygous for endogenous Ig heavy chain and/or endogenous Ig lightchain and, accordingly, incapable of producing endogenousimmunoglobulins. Regardless of the chromosomal location, an artificialIg locus of the present invention has the capacity to undergo generearrangement and thereby produce a diversified repertoire ofimmunoglobulin molecules. An Ig locus having the capacity to undergogene rearrangement is also referred to herein as a “functional” Iglocus, and the antibodies with a diversity generated by a functional Iglocus are also referred to herein as “functional” antibodies or a“functional” repertoire of antibodies.

The artificial loci used to generate such transgenic animals eachinclude multiple immunoglobulin gene segments, which include at leastone V region gene segment, one or more J gene segments, one or more Dgene segments in the case of a heavy chain locus, and one or moreconstant region genes. In the present invention, at least one of the Vgene segments encodes a germline or hypermutated human V-region aminoacid sequence. Accordingly, such transgenic animals have the capacity toproduce a diversified repertoire of immunoglobulin molecules, whichinclude antibodies having a human idiotype.

In one embodiment, the artificial loci used comprise at least onenon-human C region gene segment. Accordingly, such transgenic animalshave the capacity to produce a diversified repertoire of immunoglobulinmolecules, which include chimeric antibodies having a human idiotype.

In one embodiment, the artificial loci used comprise at least one humanC region gene segment. Accordingly, such transgenic animals have thecapacity to produce a diversified repertoire of immunoglobulinmolecules, which include antibodies having a human idiotype and a humanconstant region.

In one embodiment, the artificial loci used comprise at least oneartificial constant region gene. For example, an exemplary artificial Cconstant region gene is a constant region gene encoding a human IgG CH1domain and rat IgG CH2 and CH3 domain. Accordingly, such transgenicanimals have the capacity to produce a diversified repertoire ofimmunoglobulin molecules, which include antibodies having a humanidiotype and an artificial constant region comprising both human andnon-human components.

The transgenic vector containing an artificial Ig locus is introducedinto the recipient cell or cells and then integrated into the genome ofthe recipient cell or cells by random integration or by targetedintegration.

For random integration, a transgenic vector containing an artificial Iglocus can be introduced into a recipient cell by standard transgenictechnology. For example, a transgenic vector can be directly injectedinto the pronucleus of a fertilized oocyte. A transgenic vector can alsobe introduced by co-incubation of sperm with the transgenic vectorbefore fertilization of the oocyte. Transgenic animals can be developedfrom fertilized oocytes. Another way to introduce a transgenic vector isby transfecting embryonic stem cells or other pluripotent cells (forexample primordial germ cells) and subsequently injecting thegenetically modified cells into developing embryos. Alternatively, atransgenic vector (naked or in combination with facilitating reagents)can be directly injected into a developing embryo. Ultimately, chimerictransgenic animals are produced from the embryos which contain theartificial Ig transgene integrated in the genome of at least somesomatic cells of the transgenic animal. In another embodiment, thetransgenic vector is introduced into the genome of a cell and an animalis derived from the transfected cell by nuclear transfer cloning.

In a preferred embodiment, a transgene containing an artificial Ig locusis randomly integrated into the genome of recipient cells (such asfertilized oocyte or developing embryos). In a preferred embodiment,offspring that are nullizygous for endogenous Ig heavy chain and/or Iglight chain and, accordingly, incapable of producing endogenousimmunoglobulins and capable of producing transgenic immunoglobulins areobtained.

For targeted integration, a transgenic vector can be introduced intoappropriate recipient cells such as embryonic stem cells, otherpluripotent cells or already differentiated somatic cells. Afterwards,cells in which the transgene has integrated into the animal genome canbe selected by standard methods. The selected cells may then be fusedwith enucleated nuclear transfer unit cells, e.g. oocytes or embryonicstem cells, cells which are totipotent and capable of forming afunctional neonate. Fusion is performed in accordance with conventionaltechniques which are well established. See, for example, Cibelli et al.,Science (1998) 280:1256; Zhou et al. Science (2003) 301: 1179.Enucleation of oocytes and nuclear transfer can also be performed bymicrosurgery using injection pipettes. (See, for example, Wakayama etal., Nature (1998) 394:369.) The resulting cells are then cultivated inan appropriate medium, and transferred into synchronized recipients forgenerating transgenic animals. Alternatively, the selected geneticallymodified cells can be injected into developing embryos which aresubsequently developed into chimeric animals.

In one embodiment, a meganuclease is used to increase the frequency ofhomologous recombination at a target site through double-strand DNAcleavage. For integration into a specific site, a site specificmeganuclease may be used. In one embodiment, a meganuclease targeting anendogenous Ig locus is used to increase the frequency of homologousrecombination and replacement of an endogenous Ig locus, or partsthereof with an artificial Ig locus, or parts thereof. In oneembodiment, the transgenic animal lacks a functional Ig light chainlocus and comprises an artificial Ig heavy chain locus.

Immunoglobulins Having a Human Idiotype

Once a transgenic animal capable of producing immunoglobulins having ahuman idiotype is made, immunoglobulins and antibody preparationsagainst an antigen can be readily obtained by immunizing the animal withthe antigen. “Polyclonal antisera composition” as used herein includesaffinity purified polyclonal antibody preparations.

A variety of antigens can be used to immunize a transgenic animal. Suchantigens include but are not limited to, microorganisms, e.g. virusesand unicellular organisms (such as bacteria and fungi), alive,attenuated or dead, fragments of the microorganisms, or antigenicmolecules isolated from the microorganisms.

Preferred bacterial antigens for use in immunizing an animal includepurified antigens from Staphylococcus aureus such as capsularpolysaccharides type 5 and 8, recombinant versions of virulence factorssuch as alpha-toxin, adhesin binding proteins, collagen bindingproteins, and fibronectin binding proteins. Preferred bacterial antigensalso include an attenuated version of S. aureus, Pseudomonas aeruginosa,enterococcus, enterobacter, and Klebsiella pneumoniae, or culturesupernatant from these bacteria cells. Other bacterial antigens whichcan be used in immunization include purified lipopolysaccharide (LPS),capsular antigens, capsular polysaccharides and/or recombinant versionsof the outer membrane proteins, fibronectin binding proteins, endotoxin,and exotoxin from Pseudomonas aeruginosa, enterococcus, enterobacter,and Klebsiella pneumoniae.

Preferred antigens for the generation of antibodies against fungiinclude attenuated version of fungi or outer membrane proteins thereof,which fungi include, but are not limited to, Candida albicans, Candidaparapsilosis, Candida tropicalis, and Cryptococcus neoformans.

Preferred antigens for use in immunization in order to generateantibodies against viruses include the envelop proteins and attenuatedversions of viruses which include, but are not limited to respiratorysynctial virus (RSV) (particularly the F-Protein), Hepatitis C virus(HCV), Hepatits B virus (HBV), cytomegalovirus (CMV), EBV, and HSV.

Antibodies specific for cancer can be generated by immunizing transgenicanimals with isolated tumor cells or tumor cell lines as well astumor-associated antigens which include, but are not limited to,Her-2-neu antigen (antibodies against which are useful for the treatmentof breast cancer); CD20, CD22 and CD53 antigens (antibodies againstwhich are useful for the treatment of B cell lymphomas), prostatespecific membrane antigen (PMSA) (antibodies against which are usefulfor the treatment of prostate cancer), and 17-1A molecule (antibodiesagainst which are useful for the treatment of colon cancer).

The antigens can be administered to a transgenic animal in anyconvenient manner, with or without an adjuvant, and can be administeredin accordance with a predetermined schedule.

For making a monoclonal antibody, spleen cells are isolated from theimmunized transgenic animal and used either in cell fusion withtransformed cell lines for the production of hybridomas, or cDNAsencoding antibodies are cloned by standard molecular biology techniquesand expressed in transfected cells. The procedures for making monoclonalantibodies are well established in the art. See, e.g., European PatentApplication 0 583 980 A1 (“Method For Generating Monoclonal AntibodiesFrom Rabbits”), U.S. Pat. No. 4,977,081 (“Stable Rabbit-Mouse HybridomasAnd Secretion Products Thereof”), WO 97/16537 (“Stable Chicken B-cellLine And Method of Use Thereof”), and EP 0 491 057 B1 (“Hybridoma WhichProduces Avian Specific Immunoglobulin G”), the disclosures of which areincorporated herein by reference. In vitro production of monoclonalantibodies from cloned cDNA molecules has been described byAndris-Widhopf et al., “Methods for the generation of chicken monoclonalantibody fragments by phage display”, J Immunol Methods 242:159 (2000),and by Burton, D. R., “Phage display”, Immunotechnology 1:87 (1995).

Once chimeric monoclonal antibodies with human idiotypes have beengenerated, such chimeric antibodies can be easily converted into fullyhuman antibodies using standard molecular biology techniques. Fullyhuman monoclonal antibodies are not immunogenic in humans and areappropriate for use in the therapeutic treatment of human subjects.

Antibodies of the Invention Include Heavy Chain-Only Antibodies

In one embodiment, transgenic animals which lack a functional Ig lightchain locus, and comprising an artificial heavy chain locus, areimmunized with antigen to produce heavy chain-only antibodies thatspecifically bind to antigen.

In one embodiment, the invention provides monoclonal antibody producingcells derived from such animals, as well as nucleic acids derivedtherefrom. Also provided are hybridomas derived therefrom. Also providedare fully human heavy chain-only antibodies, as well as encoding nucleicacids, derived therefrom.

Teachings on heavy chain-only antibodies are found in the art. Forexample, see PCT publications WO02085944, WO02085945, WO2006008548, andWO2007096779. See also U.S. Pat. No. 5,840,526; U.S. Pat. No. 5,874,541;U.S. Pat. No. 6,005,079; U.S. Pat. No. 6,765,087; U.S. Pat. No.5,800,988; EP 1589107; WO 9734103; and U.S. Pat. No. 6,015,695.

Pharmaceutical Compositions

In a further embodiment of the present invention, purified monoclonal orpolyclonal antibodies are admixed with an appropriate pharmaceuticalcarrier suitable for administration to patients, to providepharmaceutical compositions.

Patients treated with the pharmaceutical compositions of the inventionare preferably mammals, more preferably humans, though veterinary usesare also contemplated.

Pharmaceutically acceptable carriers which can be employed in thepresent pharmaceutical compositions can be any and all solvents,dispersion media, isotonic agents and the like. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of the antibodiescontained therein, its use in the pharmaceutical compositions of thepresent invention is appropriate.

The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers.Examples of carriers include oils, water, saline solutions, alcohol,sugar, gel, lipids, liposomes, resins, porous matrices, binders,fillers, coatings, preservatives and the like, or combinations thereof.

Methods of Treatment

In a further aspect of the present invention, methods are provided fortreating a disease in a vertebrate, preferably a mammal, preferably aprimate, with human subjects being an especially preferred embodiment,by administering a purified antibody composition of the inventiondesirable for treating such disease.

The antibody compositions can be used to bind and neutralize or modulatean antigenic entity in human body tissues that causes or contributes todisease or that elicits undesired or abnormal immune responses. An“antigenic entity” is herein defined to encompass any soluble or cellsurface bound molecules including proteins, as well as cells orinfectious disease-causing organisms or agents that are at least capableof binding to an antibody and preferably are also capable of stimulatingan immune response.

Administration of an antibody composition against an infectious agent asa monotherapy or in combination with chemotherapy results in eliminationof infectious particles. A single administration of antibodies decreasesthe number of infectious particles generally 10 to 100 fold, morecommonly more than 1000-fold. Similarly, antibody therapy in patientswith a malignant disease employed as a monotherapy or in combinationwith chemotherapy reduces the number of malignant cells generally 10 to100 fold, or more than 1000-fold. Therapy may be repeated over anextended amount of time to assure the complete elimination of infectiousparticles, malignant cells, etc. In some instances, therapy withantibody preparations will be continued for extended periods of time inthe absence of detectable amounts of infectious particles or undesirablecells.

Similarly, the use of antibody therapy for the modulation of immuneresponses may consist of single or multiple administrations oftherapeutic antibodies. Therapy may be continued for extended periods oftime in the absence of any disease symptoms.

The subject treatment may be employed in conjunction with chemotherapyat dosages sufficient to inhibit infectious disease or malignancies. Inautoimmune disease patients or transplant recipients, antibody therapymay be employed in conjunction with immunosuppressive therapy at dosagessufficient to inhibit immune reactions.

Examples

In mice transgenic for human immunoglobulin (Ig) loci, suboptimalefficacy in delivery of fully human antibodies has been attributed toimperfect interaction between the constant regions of human membrane IgHchains and the mouse cellular signaling machinery. To obviate thisproblem, we here describe a humanized rat strain (OmniRat™) carrying achimeric human/rat IgH locus [comprising 22 human V_(H)s, all human Dand J_(H) segments in natural configuration but linked to the rat C_(H)locus] together with fully human light-chain loci [12 Vκs linked toJκ-Cκ and 16 Vλs linked to Jλ-Cλ]. The endogenous rat Ig loci weresilenced by designer zinc finger nucleases. Following immunization,OmniRats perform as efficiently as normal rats in yielding high affinityserum IgG. Monoclonal antibodies, comprising fully human variableregions with sub-nanomolar antigen affinity and carrying extensivesomatic mutations, are readily obtainable—similarly to the yield ofconventional antibodies from normal rats.

Materials and Methods

Construction of Modified Human Ig Loci on YACs and BACs

a) IgH Loci

The human IgH V genes were covered by 2 BACs: BAC6-VH3-11 containing theauthentic region spanning from VH4-39 to VH3-23 followed by VH3-11(modified from a commercially available BAC clone 3054M17 CITB) and BAC3containing the authentic region spanning from VH3-11 to VH6-1 (811L16RPCI-11). A BAC termed Annabel was constructed by joining rat CH regiongenes immediately downstream of the human VH6-1-Ds-JHs region (FIG. 1).All BAC clones containing part of the human or rat IgH locus werepurchased from Invitrogen.

Both BAC6-VH3-11 and Annabel were initially constructed in S. cerevisiaeas circular YACs (cYACs) and further checked and maintained in E. colias BACs. Construction details can be found at www.ratltd.net.

Unlike YACs, BAC plasmid preps yield large quantities of the desiredDNA. To convert a linear YAC into a cYAC or to assemble DNA fragmentswith overlapping ends into a single cYAC in S. cerevisiae, which canalso be maintained as a BAC in E. coli, two self-replicating S.cerevisiae/E. coli shuttle vectors, pBelo-CEN-URA, and pBelo-CEN-HYGwere constructed. Briefly, S. cerevisiae CEN4 was cut out as an AvrIIfragment from pYAC-RC³⁹ and ligated to SpeI—linearised pAP599⁴⁰. Theresulting plasmid contains CEN4 cloned in between S. cerevisiae URA3 anda hygromycin-resistance expression cassette (HygR). From this plasmid,an ApaLI-BamHI fragment containing URA3 followed by CEN4 or a PmlI-SphIfragment containing CEN4 followed by HygR was cut out, and ligated toApaLI and BamHI or HpaI and SphI doubly digested pBACBelo11 (New EnglandBiolabs) to yield pBelo-CEN-URA and pBelo-CEN-HYG.

To construct BAC6-VH3-11, initially two fragments, a 115 kb NotI-PmeIand a 110 kb RsrII-SgrAI, were cut out from the BAC clone 3054M17 CITB.The 3′ end of the former fragment overlaps 22 kb with the 5′ end of thelatter. The NotI-PmeI fragment was ligated to a NotI-BamHI YAC armcontaining S. cerevisiae CEN4 as well as TRP1/ARS1 from pYAC-RC, and theRsrII-SgrAI fragment was ligated to a SgrAI-BamHI YAC arm containing S.cerevisiae URA3, also from pYAC-RC. Subsequently, the ligation mixturewas transformed into S. cerevisiae AB1380 cells via spheroplasttransformation⁴¹, and URA+TRP+ yeast clones were selected. Clones,termed YAC6, containing the linear region from human VH4-39 to VH3-23were confirmed by Southern blot analysis. YAC6 was further extended byaddition of a 10.6 kb fragment 3′ of VH3-23, and conversion to a cYAC.The 10.6 kb extension contains the human VH3-11 and also occurs at the5′ end of BAC3. We constructed pBeloHYG-YAC6+BAC3(5′) for themodification of YAC6. Briefly, 3 fragments with overlapping ends wereprepared by PCR: 1) a ‘stuff’ fragment containing S. cerevisiaeTRP1-ARS1 flanked by HpaI sites with 5′ tail matching the sequenceupstream of VH4-39 and 3′ tail matching downstream of VH3-23 in YAC6(using long oligoes 561 and 562, and pYAC-RC as template), 2) the 10.6kb extension fragment with a 5′ tail matching the sequence downstream ofVH3-23 as described above and a unique AscI site at its 3′ end (usinglong oligoes 570 and 412, and human genomic DNA as template), and 3)pBelo-CEN-HYG vector with the CEN4 joined downstream with a homologytail matching the 3′ end of the 10.6 extension fragment and the HygRjoined upstream with a tail matching the sequence upstream of VH4-39 asdescribed above (using long oligoes 414 and 566, and pBelo-CEN-HYG astemplate). Subsequently, the 3 PCR fragments were assembled into a smallcYAC conferring HYGR and TRP+ in S. cerevisiae via homologousrecombination associated with spheroplast transformation, and this cYACwas further converted into the BAC pBeloHYG-YAC6+BAC3(5′). Finally, theHpaI-digested pBeloHYG-YAC6+BAC3(5′) was used to transform yeast cellscarrying YAC6, and through homologous recombination cYAC BAC6-VH3-11conferring only HYGR was generated. Via transformation, see below, thiscYAC was introduced as a BAC in E. coli. The human VH genes inBAC6-VH3-11 were cut out as a 182 kb AsiSI (occurring naturally in theHygR)—AscI fragment, and the VH genes in BAC3 were cut out as a ˜173 kbNotI-fragment (FIG. 1 top).

For the assembly of the C region with the VH overlap, the humanVH6-1-Ds-JHs region had to be joined with the rat genomic sequenceimmediately downstream of the last JH followed by rat Cs to yield acYAC/BAC. To achieve this, 5 overlapping restriction as well as PCRfragments were prepared; a 6.1 kb fragment 5′ of human VH6-1 (usingoligoes 383 and 384, and human genomic DNA as template), an ˜78 kbPvuI-PacI fragment containing the human VH6-1-Ds-JHs region cut out fromBAC1 (RP11645E6), a 8.7 kb fragment joining the human JH6 with the ratgenomic sequence immediately downstream of the last JH and containingpart of rat μ coding sequence (using oligos 488 and 346, and rat genomicDNA as template), an ˜52 kb NotI-PmeI fragment containing the authenticrat μ, δ and γ2c region cut out from BAC M5 (CH230-408M5) and thepBelo-CEN-URA vector with the URA3 joined downstream with a homologytail matching the 3′ end of the rat γ2c region and the CEN4 joinedupstream with a tail matching the 5′ region of human VH6-1 as described(using long oligoes 385 and 550, and pBelo-CEN-URA as template). Correctassembly via homologous recombination in S. cerevisiae was analysed byPCR and purified cYAC from the correct clones was converted into a BACin E. coli.

For the assembly of Annabel parts of the above cYAC/BAC containinghumanVH6-1-Ds-JHs followed by the authentic rat μ, δ and γ2c region, aswell as PCR fragments were used. Five overlapping fragments containedthe 6.1 kb fragment at the 5′ end of human VH6-1 as described above, an˜83 kb SpeI fragment comprising human VH6-1-Ds-JHs immediately followedby the rat genomic sequence downstream of the last JH and containingpart of rat Cμ, a 5.2 kb fragment joining the 3′ end of rat μ with the5′ end of rat γ1 (using oligos 490 and 534, and rat genomic DNA astemplate), an ˜118 kb NotI-SgrAI fragment containing the authentic ratγ1, γ2b, ε, α and 3′E IgH enhancer region cut out from BAC I8(CH230-162108), and the pBelo-CEN-URA vector with the URA3 joineddownstream with a homology tail matching the 3′ end of rat 3′E and theCEN4 joined upstream with a tail matching the 5′ end of human VH6-1 asdescribed above. There is a 10.3 kb overlap between the human VH6-1regions in both the BAC3 and Annabel. The human VH6-1-Ds-JHs followed bythe rat CH region together with the S. cerevisiae URA3 in Annabel can becut out as a single ˜183 kb NotI-fragment (see FIG. 1 top).

BAC6-VH3-11, BAC3 and Annabel were checked extensively by restrictionanalysis and partial sequencing for their authenticity.

b) IgL Loci

The human Igλ locus on a ˜410 kb YAC was obtained by recombinationassembly of a Vλ YAC with 3 Cλ containing cosmids²⁵. Rearrangement andexpression was verified in transgenic mice derived from ES cellscontaining one copy of a complete human Igλ YAC³⁸. This Igλ YAC wasshortened by the generation of a circular YAC removing ˜100 kb of theregion 5′ of Vλ3-27. The vector pYAC-RC was digested with ClaI and BspEIto remove URA3 and ligated with a ClaI/NgoMIV fragment from pAP 599containing HYG. PCR of the region containing the yeast centromere andhygromycin marker gene from the new vector (pYAC-RC-HYG) was carried outwith primers with 5′ ends homologous to a region 5′ of Vλ3-27 (primer276) and within the ADE2 marker gene in the YAC arm (primer 275). ThePCR fragment (3.8 kb) was integrated into the Igλ YAC using a highefficiency lithium acetate transformation method⁴² and selection onhygromycin containing YPD plates. DNA was prepared from the clones(Epicentre MasterPure Yeast DNA purification kit) and analysed for thecorrect junctions by PCR using the following oligos: 243+278 and Hyg endR+238. Plugs were made⁴³ and yeast chromosomes removed by PFGE (0.8%agarose (PFC) (Biorad) gel [6V/cm, pulse times of 60 s for 10 hr and 10s for 10 hr, 8° C.) leaving the circular yeast artificial chromosomecaught in the agarose block⁴⁴. The blocks were removed and digested withNrul. Briefly, blocks were preincubated with restriction enzyme bufferin excess at a 1× final concentration for 1 hr on ice. Excess buffer wasremoved leaving just enough to cover the plugs, restriction enzyme wasadded to a final concentration of 100 U/ml and the tube incubated at 37°C. for 4-5 hrs. The linearized YAC was ran out of the blocks by PFGE,cut out from the gel as a strip and purified as described below.

For the human Igκ locus 3 BACs were chosen (RP11-344F17, RP11-1134E24and RP11-156D9, Invitrogen), which covered a region over 300 kb from 5′Vκ1-17 to 3′ KDE⁴⁵. In digests and sequence analyses three overlappingfragments were identified: from Vκ1-17 to Vκ3-7 (150 kb NotI with ˜14 kboverlap), from Vκ3-7 to 3′ of Cκ (158 kb NotI with ˜40 kb overlap) andfrom Cκ to 3′ of the KDE (55 kb PacI with 40 kb overlap). Overlappingregions may generally favour joint integration when co-injected intooocytes²⁴.

Gel Analyses and DNA Purification

Purified YAC and BAC DNA was analysed by restriction digest andseparation on conventional 0.7% agarose gels⁴⁶. Larger fragments, 50-200kb, were separated by PFGE (Biorad Chef Mapper™) at 80 C, using 0.8% PFCAgaraose in 0.5% TBE, at 2-20 sec switch time for 16 h, 6V/cm, 10 mA.Purification allowed a direct comparison of the resulting fragments withthe predicted size obtained from the sequence analysis. Alterations wereanalysed by PCR and sequencing.

Linear YACs, circular YACs and BAC fragments after digests, werepurified by electro-elution using Elutrap™ (Schleicher and Schuell)⁴⁷from strips cut from 0.8% agarose gels run conventionally or frompulsed-field-gel electrophoresis (PFGE). The DNA concentration wasusually several ng/μl in a volume of ˜100 μl. For fragments up to ˜200kb the DNA was precipitated and re-dissolved in micro-injection buffer(10 mM Tris-HCl pH 7.5, 100 mM EDTA pH 8 and 100 mM NaCl but withoutSpermine/Spermidine) to the desired concentration.

The purification of circular YACs from yeast was carried out usingNucleobond AX silica-based anion-exchange resin (Macherey-Nagel,Germany). Briefly, spheroplasts were made using zymolyase or lyticaseand pelleted²⁰. The cells then underwent alkaline lysis, binding toAX100 column and elution as described in the Nucleobond method for alow-copy plasmid. Contaminating yeast chromosomal DNA was hydolyzedusing Plamid-Safe™ ATP-Dependent DNase (Epicentre Biotechnologies)followed by a final cleanup step using SureClean (Bioline). An aliquotof DH10 electrocompetent cells (Invitrogen) was then transformed withthe circular YAC to obtain BAC colonies. For microinjection, the insertDNA (150-200 kb), was separated from BAC vector DNA(˜10 kb) using afiltration step with sepharose 4B-CL⁴⁸.

Derivation of Rats and Breeding

Purified DNA encoding recombinant immunoglobulin loci was resuspended inmicroinjection buffer with 10 mM Spermine and 10 mM Spemidine. The DNAwas injected into fertilized oocytes at various concentrations from 0.5to 3 ng/μl.

Plasmid DNA or mRNA encoding ZFNs specific for rat immunoglobulin geneswere injected into fertilized oocytes at various concentrations from 0.5to 10 ng/ul.

Microinjections were performed at Caliper Life Sciences facility.Outbred SD/Hsd (WT) strain animals were housed in standard microisolatorcages under approved animal care protocols in animal facility that isaccredited by the Association for the Assessment and Accreditation forLaboratory Animal Care (AAALAC). The rats were maintained on a 14-10 hlight/dark cycle with ad libitum access to food and water. Four to fiveweek old SD/Hsd female rats were injected with 20-25 IU PMSG(Sigma-Aldrich) followed 48 hours later with 20-25 IU hCG(Sigma-Aldrich) before breeding to outbred SD/Hsd males. Fertilized1-cell stage embryos were collected for subsequent microinjection.Manipulated embryos were transferred to pseudopregnant SD/Hsd femalerats to be carried to parturition.

Multi-feature human Ig rats (human IgH, Igκ and Igλ in combination withrat J KO, κ KO and λ KO) and WT, as control, were analyzed at 10-18weeks of age. The animals were bred at Charles River under specificpathogen-free conditions.

PCR and RT-PCR

Transgenic rats were identified by PCR from tail or ear clip DNA using aGenomic DNA Mini Kid (Bioline). For IgH PCRs<1 kb GoTaq Green Master mixwas used (Promega) under the following conditions: 94° C. 2 mins,32×(94° C. 30 secs, 54-670 C (see supplemental Table 1 for primers andspecific annealing temperatures) 30 secs, 72° C. 1 min), 72° C. 2 mins.For IgH PCRs>1 kb KOD polymerase (Novagen) was used under the followingconditions: 95° C. 2 mins, 32×(95° C. 20 secs, 56-620 C (supplementaryTable 1) 20 secs, 70° C. 90 secs), 70° C. 2 mins. For Igκ and Igλ PCR,all <1 kb, the above condition were used except extension at 72° C. for50 secs.

RNA was extracted from Blood using the RiboPure Blood Kit (Ambion) andRNA extraction from spleen, bone marrow or lymph nodes used RNASpin minikit. (GE Healthcare). cDNA was made using Oligo dT and Promega ReverseTranscriptase at 42° C. for 1 hour. GAPDH PCR reactions (oligos 429-430)determined the concentration.

RT-PCRs were set up using VH leader primers with rat μCH2 or rat γCH2primers (supplementary Table 1). Amplification with GoTaq Green Mastermix were 94° C. 2 mins, 34×(94° C. 30 secs, 55-65° C. 30 secs, 72° C.50-60 secs), 72° C. 2 mins. PCR products of the expected size wereeither purified by gel or QuickClean (Bioline) and sequenced directly orcloned into pGemT (Promega).

Protein Purification

IgM was purified on anti-IgM affinity matrix (BAC B.V., Netherlands,CaptureSelect #2890.05) as described in the protocol. Similarly, humanIgκ and Igλ was purified on anti-L chain affinity matrix (CaptureSelectanti-Igκ #0833 and anti-Igλ #0849) according to the protocol.

For rat IgG purification²⁹ protein A and protein G agarose was used(Innova, Cambridge, UK, #851-0024 and #895-0024). Serum was incubatedwith the resin and binding facilitated at 0.1 M sodium phosphate pH 7for protein G and pH 8 for protein A under gentle mixing. Poly-prepcolumns (Bio-Rad) were packed with the mixture and washed extensivelywith PBS pH7.4. Elution buffer was 0.1 M Sodium Citrate pH 2.5 andneutralization buffer was 1 M Tris-HCl pH 9

Electrophoresis was performed on 4-15% SDS-PAGE and Coomassie brilliantblue was used for staining. MW standards were HyperPage PrestainedProtein Marker (#BIO-33066, Bioline).

Flow Cytometry Analysis and FISH

Cell suspensions were washed and adjusted to 5×105 cells/100 μl inPBS-1% BSA-0.1% Azide. Different B-cell subsets were identified usingmouse anti-rat IgM FITC-labelled mAb (MARM 4, Jackson ImmunoresearchLaboratories) in combination with anti-B cell CD45R (ratB220)-PE-conjugated mAb (His 24, BD biosciences) oranti-IgD-PE-conjugated mAb (MARD-3, Abd Serotec). A FACS Cantoll flowcytometer and FlowJo software (Becton Dickinson, Pont de Claix, France)was used for the analysis.

Fluorescence in situ hybridisation was carried out on fixed bloodlymphocytes using purified IgH and IgL C-region BACs as described.⁴⁹

Immunization, Cell Fusion and Affinity Measurement

Immunizations were performed with 125 μg PG in CFA, 150 μg hGHR in CFA,200 μg Tau/KLH in CFA, 150 μg HEL in CFA, 150 μg OVA in CFA at the baseof the tail and medial iliac lymph node cells were fused with mouseP3×63Ag8.653 myeloma cells 22 days later as described⁵⁰. For multipleimmunizations protein, 125 μg PG or HEL, or 100 μg hGHR or CD14 in GERBUadjuvant (www.Gerbu.com), was administered intraperitoneally as follows:day 0, day 14, day 28 and day 41 without adjuvant, followed by spleencell fusion with P3×63Ag8.653 cells 4 days later.⁴⁹

Binding kinetics were analyzed by surface Plasmon resonance using aBiacore 2000 with the antigens directly immobilized as described²³.

SUPPLEMENTARY TABLE 1 PCR* and RT-PCR** conditions to detect human IgHand IgL integration and expression Annealing Temp Primers (Tm-5)Fragment size IgH Hyg (5′ BAC6) Hyg 3′ F-459 54° C. ~400 bp V4-34 (BAC6)205-206 65° C. ~1 kb V4-28 (BAC6) 203-204 65° C. ~1 kb V3-11 (overlapBAC6- 448-461 60° C. ~500 bp BAC3) V1-8 (BAC3) 371-372 60° C. ~300 bpV4-4 (BAC3) 393-396 60° C. ~750 bp V6-1 (BAC3- 359-360 65° C. ~350 bpAnnabel) JH (Annabel) 368-369 62° C. ~250 bp μ-γ1 (Annabel) 583-535 62°C. ~3 kb Ura (3′ Annabel) 241-253 56° C. ~3 kb Igκ KDE 313-314 66° C.~600 bp cKappa 307-308 64° C. ~600 bp V4-1 333-334 60° C. ~300 bp V1-5329-330 64° C. ~400 bp V1-6 331-332 60° C. ~300 bp V3-7 309-310 66° C.~700 bp V3-15 311-312 66° C. ~500 bp Igλ V3-27 215-216 67° C. ~400 bpV3-19 213-214 67° C. ~700 bp V2-14 211-212 67° C. ~400 bp V middle168-169 65° C. ~500 bp JLambda 162-163 67° C. ~800 bp cLambda 170-17167° C. ~500 bp Enhancer 172-173 67° C. ~400 bp IgH VH1 Leader 390 65° C.↓ VH2 Leader 391 65° C. ↓ VH3 Leader 392 65° C. ↓ VH4 Leader 393 60° C.↓ VH6 Leader 394 65° C. ↓ VH4-39 Leader 761 55° C. ↓ Rat μCH2 345 ↑ ~1kb Rat □CH2 682 ↑ ~800 bp Ig□ HuVK1 Leader 400/474 63° C. ↓ HuVK3 Leader401/475 63° C. ↓ HuVK4 Leader 476 63° C. ↓ HuVK5 Leader 477 63° C. ↓ Hu□ C region 402 ↑ ~600 bp HuVL2 Leader 388/478 58° C. ↓ HuVL3 Leader398/479/480/ 58° C. ↓ 482/483/481/484 HuVL4 Leader 485 58° C. ↓ Hu □□Cregion 387 ↑ ~600 bp *For DNA extraction from ear and tail clips theGenomic DNA Mini Kit (Bioline) was used. For PCRs 1 kb or less in sizeGoTaq Green Master mix (Promega) was used under the followingconditions: 94° C. 2 mins, 32 × (94° C. 30 secs, Tm-5 (below) 30 secs,72° C. 1 min [50 sec for Igκ/λ]), 72° C. 2 mins. Annealing temperatureswere set at the lowest primer Tm-5° C.(www.sigmagenosys.com/calc/DNACalc.asp). For PCRs >1 kb KOD polymerase(Novagen) was used under the following conditions: 95° C. 2 mins, 32 ×(95° C. 20 secs, Tm-5 20 secs, 70° C. 90 secs), 70° C. 2 mins. **RNA wasextracted from Blood using the RiboPure Blood Kit (Ambion). RNAextracted from spleen, bone marrow or lymph nodes used the RNASpin minikit (GE Healthcare). cDNA was made using Oligo dT and Promega ReverseTranscriptase at 42° C. 1 hour. PCRs using the GoTaq Green Master mixwere set up as follows: 94° C. 2 mins, 34 × (94° C. 30 secs, Tm-5 30secs, 72° C. 1 min [50 sec for Igκ/λ]), 72° C. 2 mins.

Primers

Number Oligonucleotide sequence 5′-3′ 162 GGGGCCAAGGCCCCGAGAGATCTCAGG163 CACTGGGTTCAGGGTTCTTTCCACC 168 GTGGTACAGAAGTTAGAGGGGATGTTGTTCC 169TCTTCTACAAGCCCTTCTAAGAACACCTGG 170 AGCACAATGCTGAGGATGTTGCTCC 171ACTGACCCTGATCCTGACCCTACTGC 172 AAACACCCCTCTTCTCCCACCAGC 173CGCTCATGGTGAACCAGTGCTCTG 203 GCTATTTAAGACCCACTCCCTGGCA 204AAAACCTGCAGCAAGGATGTGAGG 205 GCTCCTTCAGCACATTTCCTACCTGGA 206CCATATATGGCAAAATGAGTCATGCAGG 211 CTCTGCTGCTCCTCACCCTCCTCACTCAGG 212GAGAGTGCTGCTGCTTGTATATGAGCTGCA 213 TGGCTCACTCTCCTCACTCTTTGCATAGGTT 214GATGGTTACCACTGCTGTCCCGGGAGTTAC 215 ATCCCTCTCCTGCTCCCCCTCCTCATTCTCTG 216TGATGGTCAAGGTGACTGTGGTCCCTGAGCTG 238 AACAAGTGCGTGGAGCAG 241GTACTGTTGACATTGCGAAGAGC 243 TGGTTGACATGCTGGCTAGTC 253TGTCTGGCTGGAATACACTC 275 AAATGAGCTTCAAATTGAGAAGTGACGCAAGCATCAATGGTATAATGTCCAGAGTTGTGAGGCCTTGGGGACTGTGTGCC GAACATGCTC 276CCAGCACTGTTCAATCACAGTATGATGAGCCTAATGGGAATCCCACTAGGCTAGTCTAGTCACCACATTAAAGCACGTGG CCTCTTATCG 278TGACCATTGCTTCCAAGTCC 307 GAGGAAAGAGAGAAACCACAGGTGC 308CACCCAAGGGCAGAACTTTGTTACT 309 TGTCCAGGTATGTTGAAGAATGTCCTCC 310TGGACTCTGTTCAACTGAGGCACCAG 311 GGCCTTCATGCTGTGTGCAGACTA 312CAGGTCGCACTGATTCAAGAAGTGAGT 313 TTCAGGCAGGCTCTTACCAGGACTCA 314TGCTCTGACCTCTGAGGACCTGTCTGTA 329 TCACGTGACTGTGATCCCTAGAA 330CACTGTTATGCCAACTGAACAGC 331 CGTAGCAGTCCCCATCTGTAATC 332ATGTCAGAGGAGCAGGAGAGAGA 333 CACGCCTCACATCCAATATGTTA 334ATACCCTCCTGACATCTGGTGAA 345 GCTTTCAGTGATGGTCAGTGTGCTTATGAC 346TGGAAGACCAGGAGATATTCAGGGTGTC 359 TTGCTTAACTCCACACCTGCTCCTG 360TGCTTGGAACTGGATCAGGCAGTC 368 CACCCTGGTCACCGTCTCC 369AGACAGTGACCAGGGTGCCAC 371 TGAGGAACGGATCCTGGTTCAGTC 372ATCTCCTCAGCCCAGCACAGC 383 CCTCCCATGATTCCAACACTG 384 CTCACCGTCCACCACTGCTG385 CTGTGCCACAAACATGCAAAGATAAGTTCCATGTGACAAGTCTGAACTCAGTGTTGGAATCATGGGAGGCGGCCGCGTTA TCTATGCTGTCTCACCATAG 387TGCTCAGGCGTCAGGCTCAG 388 TGCTCAGGCGTCAGGCTCAG 390 ATGGACTGGACCTGGAGGATCC391 TCCACGCTCCTGCTGCTGAC 392 ATGGAGTTTGGGCTGAGCTGG 393TGAAACACCTGTGGTTCTTCC 394 TCATCTTCCTGCCCGTGCTGG 396 GACTCGACTCTTGAGGGACG398 ATGTGGCCACAGGCTAGCTC 400 ATGAGGGTCCCCGCTCAG 401 ATGGAAGCCCCAGCTCAGC402 CCTGGGAGTTACCCGATTGG 412 GGCGCGCCAAGCATCATGTCCTACCTGGCTG 414CAAAGTACGTGGCACCTCCCTCGTCTTTCTTCCTCCTGCTCCAGCCAGGTAGGACATGATGCTTGGCGCGCCGTTATCTA TGCTGTCTCACCATAG 429CAGTGCCAGCCTCGTCTCAT 430 AGGGGCCATCCACAGTCTTC 448CTTCACTGTGTGTTCTTGGGATAC 459 GTGTAATGCTTTGGACGGTGTGTTAGTCTC 461GCATAGCGGCGCGCCAAGCATCATGTCCTACCTGGCTG 474 GACATGAGAGTCCTCGCTCAGC 475AAGCCCCAGCGCAGCTTC 476 ATGGTGTTGCAGACCCAGGTC 477 GTCCCAGGTTCACCTCCTCAG478 TCCTCASYCTCCTCACTCAGG 479 CGTCCTTGCTTACTGCACAG 480AGCCTCCTTGCTCACTTTACAG 481 CCTCCTCAYTYTCTGCACAG 482GCTCACTCTCCTCACTCTTTGC 483 CCTCCTCTCTCACTGCACAG 484 GCCACACTCCTGCTCCCACT485 ATGGCCTGGGTCTCCTTCTAC 488 ATTACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGGAAGAATGGCCTCTCCA GGTC 490CTGTCGTTGAGATGAACCCCAATGTGAG 534GGAACTGATGTGATCTCAGTCACACAGCTAATGCAAAGGTCAGCAGGCTGTTTACTGCCTGGAGGTTCATCGCCCAATTC CAAAGTCAC 535CTAGTCTGCATGGGTCTCCGCAAAC 550 CTGGTATAATCATAAGTCTCCACTTAATAGTTCTGTAGACAGAATCTTCATTTAGACTTACAGACCGCGGCCGCACCGCA GGGTAATAACTG 561GCAACCCTTCTTGCCACTCATGTCCCAGCTCTCACCATGTGACATAGCCTGTTAACAATTCGGTCGAAAAAAGAAAAGGA GAG 562AATGTTCTTAGTATATATAAACAAGCTACTCCCAATTCATAGTCAACTAAGTTAACATTCCACATGTTAAAATAGTGAAG GAG 566TTAACAGGCTATGTCACATGGTGAGAGCTGGGACATGAGTGGCAAGAAGGGTTGCCAGACTCCCCCTTTACCTCTATATC GTGTTC 570CTTAGTTGACTATGAATTGGGAGTAGCTTGTTTATATATACTAAGAACATTTGTCAGAAGCTCTTTCTTGTTTATTCCCA GTTTGC 583CATGTCCGTATGTTGCATCTGC 682 GGGAAGATGAAGACAGATG 761 TGGAGTGGATTGGGAGT

Results

The Human IgH and IgL Loci

Construction of the human Ig loci employed established technologies toassemble large DNA segments using YACs and BACs^(16, 19, 24-26). Asmultiple BAC modifications in E. coli frequently deleted repetitiveregions such as switch sequences and enhancers, a method was developedto assemble sequences with overlapping ends in S. cerevisiae as circularYAC (cYAC) and, subsequently, converting such a cYAC into a BAC.Advantages of YACs include their large size, the ease of homologousalterations in the yeast host and the sequence stability, while BACspropagated in E. coli offer the advantages of easy preparation and largeyield. Additionally, detailed restriction mapping and sequencinganalysis can be better achieved in BACs than in YACs.

Sequence analysis and digests identified gene clusters of interest andensured locus integrity and functionality to secure DNA rearrangementand switching over a wide region. The layout of the human IgH (human VH,D and JH segments followed by rat C genes), Igκ and Igλ loci aredepicted in FIG. 1a-c . As shown previously, overlapping regions maygenerally favor joint integration when co-injected into oocytes²⁴.Thereby, insertion of BAC6-VH3-11, a 182 kb AsiSI-AscI fragment, withBAC3, a 173 kb NotI fragment, and BAC3-1N12M518 (Hu-Rat Annabel), a 193kb NotI fragment, led to the reconstitution of a fully functionaltransgenic IgH loci in the rat genome. Similarly, the human Igκ locuswas integrated by homologous overlaps. The human Igλ locus was isolatedintact as a ˜300 kb YAC and also fully inserted into a rat chromosome.The integration success was identified by transcript analysis whichshowed V(D)J-C recombinations from the most 5′ to the most 3′ end of thelocus injected. Multiple copies were identified by qPCR (not shown) andit is likely that head to tail integrations occurred. In all cases,transgenic animals with single-site integrations were generated bybreeding.

Breeding to Homozygosity

The derivation of transgenic rats by DNA microinjection into oocytes,their breeding and immunization is comparable to the mouse. However, ZFNtechnology to obtain gene knock-outs has only been reportedrecently^(11, 13). Silencing of the rat IgH locus by J_(H) deletionusing ZFN KO technology has been described¹² and a manuscript describingsilencing of the rat IgL loci, targeting of Cκ and deletion of J-Cλgenes, is in preparation. We derived multiple founders with integratedhuman Ig loci and silenced endogenous Ig production; all analyzed by PCRand FISH with complete trans-locus integration selected and interbred(Table 2). Several founder rats carried low translocus copy numbers;with the rat C-gene BAC in OmniRat likely to be fully integrated in 5copies as determined by qPCR of Cμ and Cα products (not shown).Identification by FISH of single position insertion in many linesconfirmed that spreading or multiple integration of BAC mixtures wererare; an advantage for breeding to homozygosity, which was achieved.

TABLE 2 Generated rat lines: transgenic integration, knock-out and geneusage human V_(H) human human BAC6- rat C_(H) Igk Igl ZFN FISH VH3-11BAC3 (Annabel) BACs Igl YAC KO rat rat line 182 kb 173 kb 193 kb 300 kb300 kb J_(H) KO Igκ KO Igγ KO chromosome HC14 ✓ ✓ ✓ 5q22 OmniRat ✓ ✓ ✓ ✓✓ ✓ ✓ ✓ homozygous KOs LC#79 ✓ 17 LC#6.2 ✓ 6q23 #117 ✓ 6q32 #23 ✓  4 #35✓ 11

Rats carrying the individual human transloci—IgH, Igκ and Igλ—werecrossbred successfully to homozygosity with Ig locus KO rats. Thisproduced a highly efficient new multi-feature line (OmniRats™) withhuman V_(H)-D-J_(H) regions of over 400 kb containing 22 functionalV_(H)s and a rat C region of ˜116 kb. DNA rearrangement, expressionlevels, class-switching and hypermutation was very similar between thedifferent founders and comparable to wt rats. This is probably theresult of the associated rat constant region accommodating several Csand with the 3′E (enhancer control) region in authentic configuration.

B-Cell Development in the Knock-Out Background

To assess whether the introduced human Ig loci were capable ofreconstituting normal B-cell development flow cytometric analyses wereperformed. Particular differentiation stages were analyzed in spleen andbone marrow lymphocytes (FIG. 2), which previously showed a lack ofB-cell development in JKO/JKO rats¹², and no corresponding IgLexpression in κKO/κKO as well as in λKO/λKO animals (data not shown).Most striking was the complete recovery of B-cell development inOmniRats compared to wt animals, with similar numbers of B220(CD45R)⁺lymphocytes in bone marrow and spleen. IgM expression in a largeproportion of CD45R⁺ B-cells marked a fully reconstituted immune system.Size and shape separation of spleen cells was indistinguishable betweenOmniRats and wt animals and thus successfully restored in the transgenicrats expressing human idiotypes with rat C region. Moreover, the smallsIgG⁺ lymphocyte population was present in OmniRats (FIG. 2 right).

The analysis of other OmniRat lymphocyte tissues showed that they wereindistinguishable from wt controls and, for example, T-cell subsets werefully retained (data not shown), which further supports the notion thatoptimal immune function has been completely restored.

Diverse Human H- and L-Chain Transcripts

Extensive transcriptional analysis was carried out using bloodlymphocytes or spleen cells from transgenic rats with functionalendogenous Ig loci. RT-PCR from specific human V_(H) group forward to Cμor Cγ reverse primers, showed human V_(H)DJ_(H) usage. For L-chainanalysis group specific human Vκ or Vλ forward primers were used with Cκor Cλ reverse primers. The results (Table 3) showed the use of allintegrated human V_(H) genes regarded as functional²⁷ in combinationwith diverse use of D segments and all J_(H) segments.

TABLE 3 IgH V IgH D 4- 3- 3- 4- 3- 4- 3- 4- 2- 1- 3- 3- 3- 39 38 35 3433 31 30 28 26 24 23 22 11 3-9 1-8 3-7 2-5 7-4 4-4 1-3 1-2 6-1 1-1 2-2HC14 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ HC14 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ JKO/JKO IgH D 2-3 4-4 5-5 6-6 1-7 2-6 3-9 3-10 4-115-12 6-13 1-14 2-15 3-16 4-17 5-16 6-19 1-20 HC14 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓✓ ✓ ✓ HC14 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ JKO/JKO IgH D IgH J 2-21 3-22 4-235-24 6-25 1-26 7-27 1 2 3 4 5 6 HC14 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ HC14 ✓ ✓ ✓ ✓✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ JKO/JKO IgL V IgL J 3-27 3-25 2-23 3-22 3-21 3-19 2-183-16 2-14 3-12 2-11 3-10 3-0 2-8 4-3 3-1 1 2 3 Hu L ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓✓ ✓ ✓* ✓ ✓ ✓ ✓ #6.2 IgK V IgK J 1-17 1-16 3-15 1-12 3-11 1-9 1-8 3-7 1-61-5 3-3 4-1 1 2 3 4 5 Hu K #79 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

The analysis of class-switch and hypermutation (FIG. 3) in the JKO/JKObackground showed that these essential and highly desirable mechanismsare fully operative in OmniRats. Amplification of IgG switch productsfrom PBLs revealed an extensive rate of mutation (>2 aa changes) in themajority of cells, ˜80%, and in near equal numbers of γ1 and γ2bH-chains. A small percentage of trans-switch sequences, γ2a and 2c, werealso identified (FIG. 3), which supports the observation that thetranslocus is similarly active, but providing human (V_(H)-D-J_(H))s, asthe endogenous IgH locus²⁸. The number of mutated human Igγ and IgκL-chain sequences is ˜30% and thus considerably lower than IgG H-chains.The reason is the general amplification of L-chain from all producingcells rather than from IgG⁺ or differentiated plasma cells.

Ig Levels in Serum

To gain unambiguous information about antibody production we comparedquality and quantity of serum Ig from OmniRats and normal wt animals.Purification of IgM and IgG separated on SDS-PAGE under reducingconditions (FIG. 4) showed the expected size—˜75 kDa for μ, ˜55 kDa forγ H-chains, and ˜25 kDa for L-chains—which appeared indistinguishablebetween OmniRats and wt animals. The Ig yield from serum was determinedto be between 100-300 μg/ml for IgM and 1-3 mg/ml for IgG for both,several OmniRats and wt animals. However, as rat IgG purification onprotein A or G is seen as suboptimal²⁹, rat Ig levels may be underrepresented. Taken into consideration that these young (˜3 months old)rats were housed in pathogen-free facilities and had not been immunized,this compares well with the IgM levels of 0.5-1 mg/ml and IgG levels ofseveral mgs/ml reported for rats kept in open facilities^(30, 31).Interestingly, we were able to visualize class-specific mobility of ratIgG isotypes on SDS-PAGE as demonstrated for monoclonals²⁹. In IgGseparations (FIG. 4b ) a distinct lower yH-chain band is visible in wtbut not OmniRat Ig. This band has been attributed to γ2a H-chains, whichare not present in the OmniRat (HC14) translocus. As the IgG levels aresimilar between OmniRats and wt animals we assume class-switching issimilarly efficient. The reason that the lack of Cγ2a in OmniRats is notlimiting may be that several copies of the transgenic locus favorablyincrease the level of switch products. Purification of human Igκ and Igλby capturing with anti-L-chain was also successful (FIGS. 4c and d ) andpredicted H- and L-chain bands were of the expected size. Confirmationof the IgM/G titers was also obtained by ELISA, which determined wt andOmniRat isotype distribution and identified comparable amounts of IgG1and IgG2b (not shown).

A direct comparison of human Ig L-chain titers in solid phase titrations(FIGS. 4e and f ) revealed 5-10 fold lower levels in OmniRats than inhuman serum. However, this was expected as human control serum frommature adults can sometimes contain over 10-times higher Ig levels thanin children up to their teens³², which would be similar to the human Igκand Igλ titers in young rats. Interestingly, wt rats produce very littleendogenous Igλ while transgenic rats can efficiently express both typesof human L-chain, Igκ and Igλ.

Fully Human Antigen-Specific IgG

Several cell fusions were carried out, using either a rapidone-immunization scheme and harvesting lymph nodes or, alternatively,using booster immunizations and spleen cells (Table 2). For example, aconsiderable number of stable hybridomas were obtained after oneimmunization with human progranulin (PG) and myeloma fusion 22 dayslater. Here cell growth was observed in ˜3,520 and ˜1,600 wells in SDcontrol and OmniRat hybridoma clones, respectively. Anti-progranulinspecific IgG, characterized by biosensor measurements, was produced by148 OmniRat clones. Limiting dilution, to exclude mixed wells, andrepeat affinity measurements revealed that OmniRat clones retain theirantigen specificity. A comparison of association and dissociation ratesof antibodies from SD and OmniRat clones showed similar affinitiesbetween 0.3 and 74 nM (Table 4 and data not shown). Single immunizationswith human growth hormone receptor (hGHR), TAU receptor coupled tokeyhole limpet hemocyanin (TAU/KLH), hen egg lysozyme (HEL) or ovalbumin(OVA), followed by lymph node fusions also produced many high affinityhuman antibodies often at similar numbers compared to wt.

Furthermore, conventional booster immunizations with human PG, hGHR,human CD14 and HEL resulted in high affinities (pM range) of IgG withhuman idiotypes. OmniRats always showed the expected 4- to 5-log titerincrease of antigen-specific serum IgG, similar to and as pronounced aswt rats (Table 4a). Although the results could vary from animal toanimal, comparable numbers of hybridomas producing antigen-specificantibodies with similarly high affinities were obtained from wt animals(SD and other strains) and OmniRats. A summary of individual IgGproducing lymph node and spleen cell fusion clones, showing theirdiverse human V_(H)-D-J_(H), human Vκ-Jκ or Vλ-Jγ characteristics andaffinities are presented in Table 4b. The immunization and fusionresults showed that affinities well below 1 nM (determined by biosensoranalysis) were frequently obtained from OmniRats immunized with PG,CD14, Tau, HEL and OVA antigens. In summary, antigen-specific hybridomasfrom OmniRats could be as easily generated as from wt animals yieldingnumerous mAbs with sub-nanomolar affinity even after a singleimmunization.

TABLE 4a Diverse antigen-specific rat IgG hybridomas with fully humanidiotypes^(a) Animal Antigen Cells* fusions titer hybrids IgGs** Kd***SD PG LN 1 38400 3520 38 0.3-1.0 nM OmniRat PG LN 1 12800 1600 148 0.7-2.4 nM SD PG SP 1 51200 8000 29 ND OmniRat PG SP 1 51200 36000  24ND OmniRat hGHR LN 3 4800  704-1024 18, 3, 2 ND SD hGHR SP 1 20480053760  230  <0.07-0.4 nM   OmniRat hGHR SP 1 76800 53760   7 0.16-2.4nM  OmniRat CD14 SP 2 102400 2800-3500 54, 14 <0.1-0.2 nM  SD TAU/K LN 120000 1728  99# 0.6-2.4 nM LH OmniRat TAU/K LN 1 4800 1880 118# 0.5-3.2nM LH SD HEL LN 1 12800 1564 26 0.02-0.1 nM  OmniRat HEL LN 3 25600288-640 0, 2, 7 0.6-1.5 nM SD HEL SP 1 6400 30720   0 ND SD OVA LN 19600 1488 10 1.1-4.8 nM OmniRat OVA LN 4 8000  512-2240 0, 30, 0.7-1.5nM 0, 1 *cell numbers were 3-9 × 10⁷ per fusion **antigen specificityconfirmed by biosensor analysis ***range of 5 highest affinities #8 mAbswere specific for Tau-peptide

TABLE 4b Fusion AA Antigen Cells{circumflex over ( )} Clone K_(D) (nM)IGHV AA changes IGHD IGHJ CDR3 IGκ/λV changes IGκ/λJ PG LN 8080.1B2 0.74-31 2 7-27 3 CATGTGEDAFDIW LV3-10 1 2 or 3 PG LN 8080.2B3 1.4 3-23 13-3  4 CAKGIGSLITPPDYW LV3-19 2 2 or 3 hGHR LN 9046.8A3 2.4 1-2  6 6-193 CARVGQWLNAFDIW LV2-14 9 2 or 3 hGHR LN 9046.6E10 4.2 1-2  7 3-16 4CARRGDGAFDYW LV2-23 5 2 or 3 TAU/KLH LN 8898.2B10 0.8 4-39 5 3-22 4CARHRYYYDSRGYFIFDYW KV4-1 0 2 OVA LN 9477.2F4 2.7 3-23 6 1-26 4CAKEWGYGGSYPFDYW KV1-17 1 5 OVA LN 9477.2A9 3.9 3-11 5 3-10 4CARAYYYGSGSSLFDYW KV1-6 12 4 HEL SP 1H2 0.9 3-23 15 6-19 4CAKREYSSDWYPFDHW KV3-11 1 2 HEL SP 3C10 0.8 6-1  1 6-19 1CAREGSSGWYGFFQHW KV1-5 0 5 β-gal SP 5005.6C1 nd 6-1  5 2-21 4CARTPRLGLPFDYW KV1-12 0 4 ^(a)OmniRats (HC14/Huκ and/orHuλ/JKOJKO/KKOKKO) and control SD rats were immunized with humanprogranulin (PG), human growth hormone receptor (hGHR), human CD14,Tau-peptide (TAU-KLH), hen egg lysozyme (HEL), ovalbumin (OVA) orβ-galactosidase (β-gal). *Lymph nodes (LN) or spleen cells (SP) werefused after single or multiple administration of antigen, respectively.

Discussion

A combination of human and rat genes to assemble a novel IgH locus hasresulted in highly efficient near normal expression of antibodies withhuman idiotypes. Moreover, integration of the human Igκ and Igλ locirevealed that chimeric Ig with fully human specificity is readilyproduced and that association of rat C-regions with human L-chains isnot detrimental. Advantages of using part of the rat IgH locus are thatspecies-specific C regions and enhancer control elements are kept intheir natural configuration, with essentially only the diverse humanV_(H) D J_(H) region being transplanted. Furthermore, expression ofantibodies with rat Fc-regions allow normal B-cell receptor assembly andoptimal activation of the downstream signaling pathway essential for theinitiation of highly efficient immune responses. In particular, thequality of an immune response to antigen challenge relies on combinedactions of many receptor associated signaling and modifier components(see:

www.biocarta.com/pathfiles/h_bcrpathway.asp).

The approach of using YACs and BACs, and interchanging between the two,has the advantage of both, speed and the ability to check integrity whenmaking constructs of large regions by overlapping homology. Severalfounder rats carried low translocus copy numbers; with the rat C-geneBAC in OmniRat likely to be fully integrated in 5 copies as determinedby qPCR of Cμ and Cα products (not shown). Identification by FISH ofsingle position insertion in many lines (see Table 1d) confirmed thatspreading or multiple integration of BAC mixtures were rare; anadvantage for breeding to homozygosity, which was achieved. Little wasknown whether extensive overlapping regions would integrate, such as tomaintain the full functionality, essential for DNA rearrangement.Previously, overlapping integration has been reported but for muchsmaller regions (<100 kb)^(24, 33) and our results suggest that desiredintegration by homology or in tandem is a frequent event. This eases thetransgenic technology substantially as no laborious integration of largeYACs into stem cells and subsequent animal derivation therefrom has tobe performed^(18, 19). In addition, ZFN technology, also performed viaDNA injection^(11, 12), produced Ig KO strains easily and may well bethe future technology of choice for gene disruptions and replacement.Silenced endogenous Ig gene expression in OmniRats, containing human-ratIgH and human IgL loci, has the advantage that no interfering orundesired rat Ig could give rise to mixed products. Interestingly,immunization and hybridoma generation in OmniRats still producing wt Igrevealed that many products were fully human, human-rat IgH and humanIgL, despite incomplete Ig KOs. Here, despite the extensive number of wtV genes, it was remarkable that the introduced human genes amplifiedreadily and thus showed to be efficient expression competitors. This isin line with the observation of generally good expression levels of allour integrated transgenes, which favorably compete with the endogenousloci. Previously in mice expressing a human antibody repertoire, Ig KOswere essential as little expression of human products was found when wtIg is released^(8, 18).

It is possible that the production of fully human Ig loci even in Ig KOmice is suboptimal as strain specific cis-acting sequences are requiredfor high-level expression. In the mouse an enhancer region downstream ofCα plays a vital role in class-switch recombination³⁴ and it is likelythat elements in that region may facilitate hypermutation²³. This may bethe reason why immune responses and generation of diverse hybridomas athigh frequency may be difficult in mice carrying even a large fullyhuman locus^(35, 36). As the chimeric human-rat IgH locus facilitatesnear wt differentiation and expression levels in OmniRats, it can beconcluded that the endogenous rat C region and indeed the ˜30 kbenhancer sequence 3′ of Cα are providing optimal locus control toexpress and mature human V_(H) genes. Another region, Cδ with its 3′control motif cluster²⁶, has been removed from the chimeric C-region BACsince silencing or a lack of IgD did not appear to reduce immunefunction^(37 and refs therein). Normally, mature IgM⁺IgD⁺ B-cellsdown-regulate IgD upon antigen contact, which initiates class-switchrecombination³⁷. Thus, switching may be increased without IgD control,which is supported by our finding that IgG transcripts and serum levelsare significantly lower when the Cδ region is retained in transgenicconstructs (data not shown).

The production of specific IgG in OmniRats is particularly encouragingas we found that in various immunizations mAbs with diversity insequence and epitope, comparable to what was produced in wt controls,could be isolated via spleen and lymph node fusion. V-gene, D and Jdiversity was as expected and nearly all segments were found to be usedproductively as predicted²⁷. This was in stark contrast to mice carryingfully human transloci where clonal expansion from a few precursorB-cells produced little diversity²³. Since the number of transplantedV-genes is only about half of what is used in humans we anticipated tofind restricted immune responses and limited diversity when comparingOmniRats with wt animals. However, this was not the case and acomparison of CDR3 diversity in over 1000 clones (sequences can beprovided) revealed the same extensive junctional differences in OmniRatsas in wt animals. The few identical gene-segment combinations werefurther diversified by N-sequence additions or deletion at the V_(H) toD and/or D to J_(H) junctions and also by hypermutation. Thus, it isclear that the rat C region sequence is highly efficient in controllingDNA rearrangement and expression of human V_(H)DJ_(H). Extensivediversity was also seen for the introduced human Igκ and Igλ loci,similar to what has previously been shown in mice^(22, 23, 38). Hence,substantially reduced efficiency in the production of human antibodiesfrom mice⁷ has been overcome in OmniRats, which diversify rearrangedH-chains reliably and extensively by class-switch and hypermutation toyield high affinity antibodies in bulk rather than occasionally. Theyield of transgenic IgG and the level of hypermutation, impressivelyutilized in antigen-specific mAbs, showed that clonal diversificationand production level are similar between OmniRats and wt animals.Routine generation of high affinity specificities in the subnanomolarrange was even accomplished by different single immunizations and againcompares favorably with wt animals; results that have not been shown intransgenic mice producing human antibody repertoires from entirely humanloci¹⁸.

In summary, to maximize human antibody production an IgH locus that useshuman genes for antibody specificity but rodent genes for control ofdifferentiation and high expression should be regarded essential.L-chain flexibility is a bonus as it permits highly efficient humanIgH/IgL assembly even when wt Ig is present. For therapeuticapplications chimeric H-chains can be easily converted into fully humanAbs by C-gene replacement without compromising the specificity.

All patents and patent publications referred to herein are herebyincorporated by reference.

Certain modifications and improvements will occur to those skilled inthe art upon a reading of the foregoing description. It should beunderstood that all such modifications and improvements have beendeleted herein for the sake of conciseness and readability but areproperly within the scope of the following claims.

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1. A chimeric polynucleotide comprising at least one humanimmunoglobulin (Ig) joining (J) region gene, an Ig constant region gene,and a rat 3′ enhancer.
 2. The chimeric polynucleotide of claim 1,wherein said rat 3′ enhancer comprises he sequence set forth as SEQ IDNO:1.
 3. The chimeric polynucleotide of claim 1, further comprising atleast one human Ig variable (V) region gene and/or a human Ig diversity(D) region gene.
 4. The chimeric polynucleotide of claim 1, wherein theconstant region gene is selected from the group consisting of a humanconstant region gene and a rat constant region gene.
 5. The chimericpolynucleotide of claim 1, wherein the constant region gene comprises arat constant region gene.
 6. The chimeric polynucleotide of claim 1,wherein the constant region gene comprises a constant region geneselected from the group consisting of Cμ and Cγ.
 7. The chimericpolynucleotide of claim 1, comprising a nucleic acid sequencesubstantially homologous to bacterial artificial chromosome (BAC)Annabel, or a portion thereof.
 8. The chimeric polynucleotide of claim3, wherein said human Ig V region comprises at least one human V regiongene isolatable from BAC6-V_(H)3-11 and/or BAC3.
 9. The chimericpolynucleotide of claim 1, comprising nucleic acid sequences (a) and (b)in 5′ to 3′ order: (a) a human Ig variable region comprising human Vregion genes in natural configuration isolatable from BAC6-V_(H)3-11and/or BAC3; and (b) a human Ig joining region comprising human J regiongenes in natural configuration isolatable from the bacterial artificialchromosome (BAC) Annabel.
 10. The chimeric polynucleotide of claim 3,wherein each of the human immunoglobulin variable region, the humanimmunoglobulin diversity region, the human immunoglobulin joiningregion, the immunoglobulin constant region, and the rat 3′ enhancer arein the relative positions shown in FIG. 1 a.
 11. The chimericpolynucleotide of claim 10, comprising a nucleic acid sequencesubstantially homologous to the nucleic acid sequence set forth as SEQID NO:2.
 12. The chimeric polynucleotide of claim 10, comprising anucleic acid sequence substantially homologous to the nucleic acidsequence set forth as SEQ ID NO:11.
 13. The chimeric polynucleotide ofclaim 3, wherein said V-D-J regions are rearranged and form a completeexon encoding a heavy chain variable domain.
 14. A rodent cellcomprising the chimeric polynucleotide of claim
 1. 15. The rodent cellof claim 14, further comprising a polynucleotide encoding a functionalimmunoglobulin comprising V-, J- and constant region genes and a nucleicacid sequence substantially homologous to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7.